The induction of interferon (IFN) plays a critical role in the antiviral innate immune response. IFNs are classified into type I and type II subgroups according to sequence homologies and receptor specificities. The type I IFNs, which have especially strong antiviral activities and bind to the type I IFN receptor (IFNAR), include IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ. The best studied of the type I IFNs are IFN-α and IFN-β. These IFNs are produced in response to the sensing of pathogen-associated molecular patterns (PAMPs), such as viral double-stranded RNA, by pattern recognition receptors. IFNs are secreted from the infected cells and bind to IFNAR on nearby cells to initiate signaling cascades that enhance cellular resistance to viral infection [extensively reviewed in (Samuel, 2001)]. IFN-α therapy has been successful in treating viral infections in people, most notably for hepatitis C with the current standard of care for chronic hepatitis C being a combination of IFN-α and the antiviral drug ribavirin (Fried et al., 2002). Recent advances in IFN-α therapy for such viral infections include the addition of a branched 40 kDa polyethylene glycol molecule (pegylation) to synthetic IFN-α, which enhances the effective half-life of the IFN when compared to its native form. Significant improvement was also obtained with a 166 amino acid synthetic, highly potent IFN-α (IFN-alfacon-1), which was derived by comparing predicted amino acid sequences of several natural IFN-α subtypes and assigning the most frequently observed amino acid in each corresponding position resulting in a consensus sequence. Both types of synthetic IFN are now manufactured using bacterial expression methods.
Although not tested in humans, IFN treatment against viruses considered potential bioterrorism or biowarfare threats such as Venezuelan equine encephalitis virus (VEEV) (Lukaszewski and Brooks, 2000), Rift Valley fever virus (RVFV) (Morrill et al., 1989), and Ebola virus (EBOV) (Bray, 2001; Jahrling et al., 1999) has shown efficacy in animal studies. To counteract the potent antiviral effects of IFN these viruses, as well as most or all other viruses, have developed mechanisms to antagonize multiple steps leading to IFN induction or signaling. For example, the VP35 protein of EBOV antagonizes the IFN response by disrupting signaling from the IFN promoter as well as by blocking the phosphorylation and activation of PKR (protein kinase R) (Basler et al., 2000; Feng et al., 2007; Harcourt et al., 1999). In addition, EBOV VP24 counteracts IFN signaling by binding to karyopherin-α nuclear localization signal receptors, thus preventing nuclear localization of the transcription factor STAT1 (signal transducer and activator of transcription 1) (Mateo et al.). For RVFV, a nonstructural protein, NSs, was found to both suppress the transcription of host mRNAs, including IFN mRNAs, and to induce post-transcriptional downregulation of PKR. (Billecocq et al., 2004; Bouloy et al., 2001; Habjan et al., 2009; Ikegami et al., 2009). For VEEV, disruption of tyrosine phosphorylation and nuclear translocation of STAT1 and STAT2 in response to IFN signaling was shown to correlate with expression of nonstructural, but not structural proteins (Simmons et al., 2009; Yin et al., 2009).
Currently, the treatment options for infection with potential biowarfare/bioterrorism viruses are extremely limited. IFNs, especially those that could overcome viral IFN antagonism mechanisms, could provide a prophylactic or therapeutic countermeasure for infection, perhaps in combination with novel antiviral drugs. Attempts to generate enhanced IFN include not only consensus sequence genes, but also genes derived through technologies that randomly blend related genes (gene shuffling) to create unique hybrid expression products with altered biological activities. In studies by others, gene shuffling was used to blend human IFN-α genes to create proteins with enhanced cross-species antiviral activity (Chang et al., 1999). One blended IFN-α was reported to have a 285,000-fold increase in activity compared to human IFN-α2a and a 185-fold increase over human IFN-α1 against encephalomyocarditis virus (EMCV) infection in a cytopathic effect reduction assay with mouse L929 cells (Chang et al., 1999). In another study, gene shuffling was used to derive IFN-α proteins that exhibited superior antiviral potency as well as reduced antiproliferative activity relative to IFN-alfacon-1 (Brideau-Andersen et al., 2007).